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Expanded genome modification roles for CRISPR/Cas9 using Cas9-VPR and shortened sgRNAs

gBlocks® Gene Fragments used in synthetic genetic circuit demonstrating new genome-editing capabilities of Cas9

Kiani S, Chavez A, et al. (2015) Cas9 gRNA engineering for genome editing, activation and repression. Nat Methods. 2015 Sep 7 [Epub ahead of print]

Background

The CRISPR/Cas9 system has revolutionized synthetic biology research because of its quick, cost-effective genome editing capabilities. Although its mechanism is now well-understood, researchers are investigating how slight changes to the system can make it an even more powerful tool for genome modification. In this paper, the authors fuse a transcriptional activator, VPR, to Cas9, and introduce a CRISPR-based method for controlling Cas9 function by varying sgRNA protospacer lengths. The technique allows a single Cas9 enzyme to perform genome editing and gene regulation functions simultaneously, and the authors show how this can be used to construct complex genetic circuits.

Experiment

The scientists first targeted Cas9-VPR with shortened sgRNA protospacers to a fluorescent reporter that was transfected into human cells, and quantified deletion or activation of the reporter using qPCR. They then targeted Cas9-VPR, with 14 and 20 nt sgRNA protospacer elements, to endogenous genes and measured resulting expression levels and mutation rates. Next, Kiani and colleagues evaluated Cas9-VPR for its ability to achieve simultaneous transcriptional activation and repression within a single cell using a 14 nt protospacer element. The group used IDT gBlocks® Gene Fragments to assemble the U6-driven sgRNA expression cassettes and a CRISPR-repressible promoter (CRP) library.

Results

The researchers found that Cas9-VPR had similar nuclease activity to a wild type Cas9 enzyme when used with a 20 nt protospacer element and targeted to the fluorescent reporter. However, when used with a 14 nt protospacer element, Cas9-VPR activated gene expression comparably to nuclease-null Cas9-VPR. These findings led the researchers to target Cas9-VPR to endogenous genes in a population of cells, using both protospacer lengths. In doing so, they found that the 14 nt protospacer element increased expression of TTN and MIAT, while the 20 nt protospacer element induced mutation in ACTC1. Finally, the scientists’ flow cytometry analysis demonstrated that by combining Cas9-VPR with an sgRNA bearing a 14 nt protospacer element, they achieved both transcriptional activation and transcriptional repression of fluorescent proteins EYFP and tdTomato within a single cell.

These results confirm that sgRNA protospacer length has profound effects on the CRISPR system and show that when fused with VPR, Cas9 can be an incredibly versatile tool for multiple forms of genome modification. These observations are demonstrated in this paper, as the authors successfully construct a genetic circuit that utilizes all of the new-found genomic functions of Cas9-VPR: cleavage, activation, and repression.

Product Focus: gBlocks® Gene Fragments

gBlocks® Gene Fragments are 125–2000 bp, double-stranded, sequence-verified, DNA genomic blocks that can be shipped in 2–5 working days for affordable and easy gene construction or modification. gBlocks Gene Fragments have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards. With gBlocks Gene Fragments, IDT provides a tool that accelerates your research and makes synthetic biology easier and more accessible than ever, for any lab.

Learn more about gBlocks Gene Fragments at www.idtdna.com/gblocks.

Author: Nolan Speicher is a scientific writing intern at IDT.

© 2015 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


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